Processing of a sample such as a biological sample for fermentation and centrifugation typically requires use of more than one vessel. For example, fermentation is carried out in one vessel and then the sample must be transferred to another vessel before the sample can be placed in a centrifuge for further processing. In addition, such multiple process procedures may require significant manual intervention to transfer the sample from one vessel to another and from one processing device or station to another.
Fermentation is a key technology in many fields and industries and is performed both on a mass production scale and on an experimental, bench top scale. For example, fermentation systems are used for the production of a large number of products such as antibiotics, vaccines, synthetic biopolymers, synthetic amino acids, and proteins. Fermentation technology is integral in the production of recombinant proteins using biological organisms such as E. coli and many other cell cultures. For example, production of commercial pharmaceuticals such as recombinant insulin (Eli Lilly), erythropoietin (Amgen), and interferon (Roche) all involve fermentation as an essential step.
Rapid advances in biotechnology have enabled the development of high throughput alternatives to traditional laboratory bench top processes. Unfortunately, fermentation methods have not been amenable to automation because limits in current fermentation technology prevent the uninterrupted processing flow that characterizes automated high throughput systems. Existing fermentation systems typically involve multiple handling steps by either a batch processing method or a continuous processing method.
Current production scale batch processes involve first fermenting in large scale, bulk fermentation vessels, then processing the fermentation medium to isolate the desired fermentation product, followed by transferring this product into the production stream for further processing, and finally cleaning the fermentation apparatus for the next batch. In a large-scale batch culture, it is generally necessary to provide a high initial concentration of nutrients in order to sustain cell growth over an extended time. As a result, substrate inhibition may occur in the early stages of cell growth and then may be followed by a nutrient deficiency in the late stages of fermentation. These disadvantages result in sub-optimal cell growth rates and fermentation yields. Another disadvantage of this method lies in the need to individually dispense the fermentation products from the bulk fermentation apparatus into separate sample vessels for further processing. Thus, by producing the fermentation product on a bulk scale, the fermentation product is not immediately available for automated processing. Further disadvantages include the decreased efficiency of both transferring the material to another sample vessel, as well as cleaning and sterilizing the fermentation apparatus for the next batch. These disadvantages result in increased production costs, inefficient production times and decreased yields.
Continuous batch processes involve siphoning off the fermentation product from the bulk fermentation vessel and continuously adding nutrients to the fermentation medium according to a calculated exponential growth curve. This curve, however, is merely an approximation that does not accurately predict cell growth in large, industrial scale quantities of fermentation medium. Consequently, due to the unpredictable nature of large-scale fermentation environments, experienced personnel are required to monitor the feeding rate very closely. Changes in the fermentation environment may result in either poisoned fermentation products being siphoned off into the production stream or sub-optimal production yields due to starved fermentation mediums. As a further disadvantage, unpredictable fermentation product yields affect the accuracy of subsequent processing steps. For example, when the fermentation yield decreases, the amount of aspirating, the amount of reagent dispensed, or the centrifuge time is no longer optimized, or even predictable. Frequent or continuous monitoring of the fermentation process and adjustment of the fermentation conditions is often not practicable or efficient in a production scale process.
Fermentation remains a key-processing step in a number of industries, particularly in biotechnology industries, and thus a need exists for incorporating fermentation processes into current multiple process systems, such as automated high throughput systems. A process that produces a precise, known, and repeatable amount of unpoisoned fermentation product with limited human interaction or sample vessel transfer is essential to integrating fermentation into modem production processes.
Centrifugation, like fermentation, is a key technology in many fields and industries. It may be performed on a mass production scale or an experimental, bench top scale. For example, centrifuges are used in a wide variety of disciplines, including the chemical, agricultural, medical and biological fields. In particular, centrifuge technology is integral to chemical syntheses, cell separations, radioactive isotope analyses, blood analyses, assaying techniques, as well as many other scientific applications.
The recent identification of the more than 31,000 genes comprising the human genome highlights one important use of centrifuge technology, namely the determination of each gene's function, which has become of paramount biomedical importance. Because certain genes encode multiple proteins, it is estimated that as many as 1,000,000 proteins must be produced and isolated to understand completely the function of each gene in the human genome. Centrifugation is an important step in isolating and separating proteins, but protein isolation frequently requires several labor intensive and time-consuming sequential procedures that often involve more than one centrifugation step for each isolation process.
Particularly for commercial applications, these proteins and other products utilizing centrifuge technology must be synthesized analyzed or isolated on a production scale. Likewise, rapid advances in laboratory equipment have transitioned traditional laboratory bench top processes to more automated high-throughput systems. Unfortunately, limits in current centrifuge technology prevent the uninterrupted processing flow that characterizes automated high throughput systems.
These and other disadvantages are highlighted in a typical protein isolation process. Generally, a sample is centrifuged, removed from the centrifuge and a portion of the sample is removed, often by aspiration, from the sample at a separate processing station. At yet another processing station, a reagent is often dispensed into the remaining sample, followed by sonication in a separate sonication device (also at another processing station). Once the contents of the sample have been sonicated, the sample is placed back in the centrifuge and undergoes another centrifugation step. Frequently, this centrifugation-aspiration-dispensing-sonication-centrifugation cycle is repeated more than once for a particular protein isolation.
This cycle and all its drawbacks are also representative of many other applications involving centrifugation. Disadvantageously, typical sonication and centrifugation steps are not amenable to automated processing flows because of the need to physically transfer large numbers of samples to and from various processing stations. For example, in the example described above, a sample must be moved from a centrifugation station to an aspirating station, to a dispensing station, to a sonication station, and back to a centrifugation station. Unfortunately, this cycle may be repeated several times before a particular protein or other targeted material is isolated. Accordingly, the labor-intensive nature of the isolation process poses severe time constraints and cost increases, particularly when integration of the centrifuge step or the sonication step into an automated multiple process system is currently unavailable.
As centrifugation remains a key processing step in a number of industries, and particularly in biotechnology industries, a critical need exists for incorporating centrifugation processes into current multiple process systems such as automated high throughput systems. Developing a method and apparatus that reduces the need to transfer samples to a separate processing station for each processing step is essential to integrating centrifugation into modem production processes such as an automated high throughput system.
Accordingly, there is a need for a method and an apparatus that can perform multiple processing steps on a sample in a single vessel accurately and precisely while minimizing waste.